Theriogenology 184 (2022) 110e123 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification Tania García-Martínez a, 1, Iris Martínez-Rodero a, 1, Joan Roncero-Carol a, nez-Ortiz a, Adam Z. Higgins b, Teresa Mogas a, *  n Ya Iva a Dept. of Animal Medicine and Surgery, Autonomous University of Barcelona, ES-08193, Cerdanyola del Vall es, Spain b School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, 97331-2702, USA a r t i c l e i n f o a b s t r a c t Article history: The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted Received 1 December 2021 reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine Received in revised form oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve 25 February 2022 and optimize methods of cryopreservation. However, these procedures employed are not always Accepted 26 February 2022 Available online 10 March 2022 designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic Keywords: Membrane permeability observations, we propose shorter dehydration-based protocols at different temperatures (25 C vs. Osmotic behavior 38.5 C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed Meiotic spindle to equilibration solution (ES) at 25 C and 38.5 C and effects of optimized exposure times for each DNA fragmentation temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA Embryo development fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide þ 7.5% ethylene glycol in TCM199 medium þ 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5 C and 5 min 30 s at 25 C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/ warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA frag- mentation were observed in the fresh control group and oocytes vitrified at 38.5 C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25 C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5 C and fresh controls, while these rates were lower in vitrified/25 C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5  C appears to improve the quality of vitrified/ warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blas- tocyst rates and embryo quality. © 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction preserving female genetically valuable material or avoiding the loss of rare genotypes in the event of an unexpected catastrophe. The Oocyte cryopreservation is today the most promising and cost- value and feasibility of genome resource banking become even effective option for the storage of female germplasm. In domestic more important for the preservation of ancient breeds, as over the animals, cryopreservation of oocytes to create banks is a way of last decades, farm animal genetic diversity has rapidly declined due * Corresponding author. E-mail addresses: taniagarciamartinez@gmail.com (T. García-Martínez), iris.martinez@outlook.com (I. Martínez-Rodero), joanroncerocarol@gmail.com (J. Roncero-Carol), ivan.yanez22@gmail.com (I. Ya nez-Ortiz), Adam.Higgins@oregonstate.edu (A.Z. Higgins), teresa.mogas@uab.cat (T. Mogas). 1 Both authors contributed equally. https://doi.org/10.1016/j.theriogenology.2022.02.024 0093-691X/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 to changing market demands and intensification of agriculture [1]. assess the optimized CPA addition procedure by examining induced Cryopreserved oocyte banks could also help improve research en- toxicity/osmotic stress and the whole vitrification process by deavors by providing accessible and repeatable supplies and analyzing oocyte spindle morphology, DNA fragmentation, and reducing the effects of seasonal variation in oocyte quality. subsequent embryo development. Despite some successes, there still remain shortcomings with methods used to cryopreserve bovine oocytes (reviewed by Refs. [2,3]). Many of the problems associated with the cryopreser- 2. Materials and methods vation of mature oocytes are related to exposure to cryoprotective agents (CPAs). The ability of CPAs to avoid ice formation increases 2.1. Chemicals and suppliers with concentration, however, so does their toxicity. CPAs can have a damaging effect on cells due to their intrinsic chemical toxicity, and All chemicals and reagents used in this study were purchased they can also produce osmotic damage, which could be defined as from Sigma Chemical Co (St. Louis, MO, USA) except where other- the creation of an osmolarity that is not tolerated by the cells [4]. wise indicated. Both types of damage are dependent on the duration and the temperature and of the exposure, so these parameters have to be 2.2. Oocyte collection and in vitro maturation carefully controlled in vitrification protocols. It is well known that both osmotic stress and temperature fluctuations below their The methods used for the in vitro maturation (IVM), in vitro physiological range directly affect the developmental potential of fertilization (IVF), and in vitro culture (IVC) of the bovine oocytes oocytes, causing disruption in cytoskeleton structures, especially have been described elsewhere [16]. Briefly, ovaries were collected depolymerization of the meiotic spindle [5,6]. from cows at a local abattoir (Escorxador Sabadell, S.A., Sabadell, Most of the currently employed vitrification protocols for bovine Spain) and immediately transported to the laboratory in pre- oocytes are time-specific and require a given treatment duration warmed (35e37 C) saline solution (0.9% NaCl). Cumulus-oocyte with an equilibration solution (ES), whose composition is similar to complexes (COCs) were obtained by aspirating 3e8 mm diameter the vitrification solution (VS) but less concentrated, and a given follicles using an 18 g needle and washed three times in modified exposure time to VS before plunging the oocytes into liquid nitro- Dulbecco's PBS (PBS supplemented with 0.036 mg/mL sodium py- gen; some require observation of oocyte volume response in the ES; ruvate, 0.05 mg/mL gentamicin, and 0.5 mg/mL bovine serum al- some demand exposure to ES and VS be conducted at room tem- bumin, BSA). Only COCs with at least three compact layers of perature (RT), biological temperatures or higher than biological cumulus cells and a homogeneous cytoplasm were used. After three temperatures (reviewed by Ref. [7]). These procedures, however, washes in PBS, groups of 50 COCs were transferred to 500 mL of rarely consider the osmotic tolerance of the bovine oocytes ac- maturation medium in four-well dishes and cultured for 24 h at cording to temperature and time of CPA addition and removal. 38.5 C in a 5% CO2 humidified air atmosphere. The maturation When this characteristic is considered, the time needed to prepare medium was composed of tissue culture medium (TCM-199) sup- oocytes for vitrification may be shortened while maintaining the plemented with 10% (v/v) fetal bovine serum (FBS), 10 ng/mL critical cytosolic solute content necessary for successful epidermal growth factor, and 50 mg/mL gentamicin. vitrification. Previous papers have already described in detail bovine oocyte membrane permeability parameters and their application for 2.3. Modeling the membrane permeability of bovine MII oocytes mathematical modeling for finding optimal cryopreservation pro- tocols [5,8e12]. These studies reported water plasma membrane 2.3.1. Measurement of oocyte volumetric changes following CPA and CPA permeability characteristics of bovine oocytes in the exposure at 25  C and 38.5  C presence of the most used CPAs such as dimethyl sulfoxide After 22 h of IVM, oocytes were denuded of cumulus cells by (Me2SO), ethylene glycol (EG), propylene glycol, as well as glycerol gentle pipetting. Only mature oocytes showing a normal appear- solutions; performance at multiple temperatures (4e38 C) with ance and a visible first polar body were used. An oocyte was placed activation energies derived; while comparing different develop- in a 25 mL-drop of holding medium (HM: TCM199-Hepes supple- mental stages (in vitro matured, fertilized oocytes); and many os- mented with 20% (v/v) FBS) covered with mineral oil, and held with motic volume response simulations/proposed cryopreservation a holding pipette (outer diameter, 100 mm) (MPHL-35, Life Global protocols. From these studies, it was obvious that EG or Me2SO group, Guilford, United States) connected to a micromanipulator on were more adequate as CPAs for bovine oocytes, compared with an inverted microscope (Zeiss Axio Vert A1, Germany). An initial glycerol [12]. For sugars, the use of trehalose, as a non-penetrating photograph was taken of the oocyte to calculate its initial volume. cryoprotectant, produced worse results compared with sucrose The oocyte was then covered with another pipette of larger inner [13]. And the combination of Me2SO and EG at lower concentrations diameter (600 mm) (G-1 Narishigue, Tokyo, Japan) connected to a with the addition of a non-penetrating CPA such as sucrose is different micromanipulator. Then, by sliding the dish, the oocyte commonly added to the standard vitrification solutions of bovine was introduced in a 25 mL drop containing 1.55 M Me2SO or oocytes to reduce the toxic effect of a high total concentration of 1.55 M EG Ref. [14] at 25 C or 38.5 C and left for 5 min. The volu- one CPA alone [14]. metric response of the oocyte during the experiments was recorded In the context of optimization of cryopreservation procedures every 5 s using a time-lapse video recorder (Zeiss Zen imaging for vitrification in the presence of combined CPAs, one goal is to software/Axiocam ERc 5s). The video was converted into image determine the best exposure time to the equilibration solution at a frames and a series of images taken at 5 s-intervals were used for specific temperature [15]. So, the present study aimed to optimize analysis. Oocyte volume was measured through its cross-sectional oocyte vitrification protocols via incorporating insights on osmotic area using ImageJ software. An average of 10e14 mature oocytes oocyte volume responses during incubation in cryopreservation that remained close to spherical in shape were individually solutions. For that purpose, we first determine both predictively analyzed for each CPA and temperature. Osmotic responses of the and experimentally the osmotic behavior of metaphase II (MII) oocyte were calculated by measuring oocyte volume changes when bovine oocytes in terms of membrane permeability parameters (Lp exposed to 1.55 M Me2SO or 1.55 M EG at the different and Ps) in response to ES CPAs at 25 C and 38.5 C, and then we temperatures. 111 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 2.3.2. Membrane permeability parameters Temperature differs between 25 C (298 K) or 38.5 C (311K) and is The experimental data were fitted to a two-parameter (2P) indicated in the first equation of the three differential equations transport formalism [17] to determine the permeability of bovine solved in Matlab software. MII oocytes to water (Lp) and solutes (Ps). The 2P model assesses The rate of Me2SO transport is given by: mass transfer dynamics through the cell over time by assuming there is no intramembrane interaction between water and dNMe2SO e i ¼ PMe2SO AðMMe2SO  MMe2SO Þ (3) permeable solutes. This model uses a pair of coupled differential dt equations to describe cell volume changes and moles of an intra- And the rate of EG transport is given by: cellular permeating solute when the cell is exposed to a ternary solution consisting of a permeable solute (CPA), an impermeable dNEG e i solute (NaCl), and solvent (water). ¼ PEG AðMEG  MEG Þ (4) dt Water flux into the cell over time is expressed as: In this study, the water and solute permeability used for the CPA dVw addition predictions at 25 C or 38.5 C were previously estimated ¼  Lp ARTðM e  M i Þ (1) (see section 2.3.2). Water permeability was assumed to be the dt average value for the individual Lp values obtained when the oo- where Vw is the cell water volume, Lp is the membrane permeability cytes were exposed to 1.55 M Me2SO or 1.55 M EG at 25 C or 38.5 C to water (hydraulic conductivity), A is the area of the plasma (2.41 and 1.64 mm/atm  min, respectively). The values for PMe2SO membrane, R is the universal gas constant, T is the absolute tem- and PEG were 0.59 and 0.78 mm/s, respectively at 25 C, and 1.38 and perature, and Me and Mi are the total external and internal osmo- 1.94 mm/s, respectively at 38.5 C. The oocyte was assumed to be a lalities, respectively. perfect sphere with a radius of 56.1 mm in isotonic medium, and an The rate of CPA transport is given by: osmotically inactive volume of 25% of its initial volume [8,12]. Predictions for the CPA addition process (equilibration solution) dNs were run at 25 C or 38.5 C for bovine MII oocytes, for which the ¼ Ps AðMse  Msi Þ (2) dt exposure time was limited to 9 min. Key parameters for oocyte survival of vitrification, such as the volumetric excursion of the where Ns is the intracellular moles of CPA, Ps is CPA permeability, oocyte and total cytoplasmic solute concentration were determined and Mis and Mes are intracellular and extracellular CPA molality, and compared between the two temperatures. respectively. Volumetric data for each oocyte at each concentration and 2.4. In vitro osmotic behavior following ES exposure at 25 C and temperature were fitted to the 2P model to determine Lp and Ps. The 38.5 C differential equations (equation (1) and (2)) were solved in Matlab software using the ode45 function, which implements an explicit To determine if the model predictions were accurate, our in Runge-Kutta formula [18,19]. To estimate permeabilities, model silico dehydration profiles obtained with the theoretical models predictions were fitted to the data by minimizing the sum of the were tested in vitro by observing the osmotic behavior of bovine MII error squared in Matlab using the fminsearch function, which im- oocytes exposed to the equilibration solution at 25 C or 38.5 C. The plements the Nelder-Mead simplex algorithm [20]. The various methodology used was the same as described in section 2.3.1 with constants and parameters appearing in the equations are listed in the difference that oocytes were exposed for 9 min to the equili- Table 1. bration solution (ES; 7.5% (v/v) EG and 7.5% (v/v) Me2SO in TCM-199 Hepes) at 25 C or 38.5 C. An average of 20 mature oocytes that 2.3.3. Prediction of cell volume changes during exposure of bovine remained close to spherical in shape were individually analyzed for oocytes to the equilibration solution at 25  C and 38.5  C each temperature. To predict the cell volume response and intracellular CPA con- centration when oocytes are exposed to ES (the first step of CPA addition), it is essential to define two solute equations (equations 2.5. Oocyte vitrification and warming (3) and (4)), one for Me2SO and another for EG. Accordingly, a system of three linear ordinary differential equations needs to be After 22 h of IVM, oocytes were vitrified/warmed as previously solved for the three variables (Vw, NMe2SO, and NEG). These three described [21]. Oocytes were partially denuded by gently pipetting differential equations (equation (1), (3) and (4)) were solved as in PBS. Based on the results obtained in the previous experiments, described above in Matlab software using the ode45 function, oocytes with only corona radiata cells were transferred to ES con- which implements an explicit Runge-Kutta formula [18,19]. taining 7.5% (v/v) Me2SO and 7.5% (v/v) EG for different periods of time depending on the temperature (see experimental design). Oocytes were then transferred to the vitrification solution (VS: 15% Table 1 (v/v) Me2SO, 15% (v/v) EG, and 0.5 M sucrose). After incubating for Constant and parameters used in 2P model. 30e40 s, up to five oocytes were loaded onto a Cryotop and almost Description Values Symbol all the solution was removed to leave only a thin layer covering the Universal gas constant 8.314 m3 Pa K1 mol1 R oocytes. Oocytes were immediately plunged into liquid nitrogen. Absolute temperature 298 K or 311 K T The entire process from exposure to the vitrification solution to Partial molar volume of water 18.02 x1012mm3mol1 yw plunging in liquid nitrogen was completed within 60 s. Partial molar volume of CPA: Warming was performed by quickly immersing the tip of the 6 1 EGa 55.8 3  10 m mol ys Cryotop in warming solution 1 (WS1: HM supplemented with 1 M Me2SOa 71.3  106 m3 mol1 Osmotically inactive volume MII oocytes): 0.25 [8,12] Vb sucrose) at 38.5 C. After 1 min, oocytes were transferred into WS2 Isotonic cell volume 7.40  105 mm3 [68] V0 (HM supplemented with 0.5 M sucrose) for 3 min and then to HM Abbreviations: CPA, cryoprotectant; EG, ethylene glycol; Me2SO, dimethyl sulfoxide; for 5 min. Oocytes were then transferred back into the maturation MII, metaphase II. medium and allowed to mature for 2 additional hours at 38.5 C in a Partial molar volumes of cryoprotectants from Vian et al. [69]. humidified air containing 5% CO2. 112 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 2.6. In vitro fertilization and embryo culture (DAPI; excitation 405 nm; emission 460 nm). The criteria used to classify chromosome and microtubule distributions have been After 24 h of IVM, oocytes were in vitro fertilized (IVF) at 38.5 C described elsewhere [24]. In brief, the meiotic spindle was defined in a 5% CO2 atmosphere and cultured as previously described by as normal when the classic symmetrical barrel shape was observed, Arcarons et al. [22]. Briefly, high motility and good morphology with chromosomes aligned regularly in a compact group along the spermatozoa from Asturian bulls (ASEAVA, Llanera, Asturias, Spain) equatorial plane. In contrast, abnormal spindles were recorded were obtained by centrifuging frozen/thawed sperm at 300g for when there was microtubule decondensation or partial or total 10 min at RT on a discontinuous gradient consisting of 1 mL of 40% disorganization, or as absent when there was a complete lack of and 1 mL of 80% BoviPure (Nidacon Laboratories AB, Go €teborg, microtubules. Chromosome organization was considered abnormal Sweden) according to the manufacturer's instructions. Viable when chromosomes were dispersed or had an aberrant, less spermatozoa collected from the bottom were washed with 3 mL of condensed appearance or lacking when chromosomes were BoviWash (Nidacon International, Go € teborg, Sweden) and pelleted missing. Detailed images of these normal and abnormal patterns by centrifugation at 300g for 5 min at RT. Spermatozoa were are shown in Fig. 1. counted in a Neubauer chamber and diluted in an appropriate volume of fertilization medium to give a final concentration of 2.8. TUNEL detection of fragmented oocyte DNA 1  106 spermatozoa/mL. One-hundred-microliter droplets of diluted sperm were prepared under mineral oil and 20 oocytes/ Oocyte DNA fragmentation was detected by terminal deoxy- droplet were co-incubated at 38.5 C, 5% CO2, and high humidity. nucleotidyl transferase (TdT) mediated dUTP-digoxigenin nick end- The fertilization medium consisted of Tyrode's medium supple- labeling (TUNEL) using a kit (in situ Cell Death Detection Kit, mented with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM Fluorescein) according to the manufacturer's instructions (as sodium pyruvate, 6 mg/mL fatty acid-free BSA and 1 mg/mL described in Vendrell-Flotats et al. [25] with some modifications). heparin-sodium salt. Briefly, after 24 h of maturation, oocytes were completely denuded At 18e20 h post-insemination, oocyte survival was evaluated of cumulus cells by gentle pipetting and fixed in 2% (w/v) para- morphologically. The criteria used to classify oocytes as surviving or formaldehyde in PBS for 30 min at 38.5 C. After three washes in PBS degenerated have been described elsewhere [23]. Briefly, oocytes with 0.3% polyvinylpyrrolidone (PVP), fixed oocytes were per- with intact oolemma, intact zona pellucida, and homogenous and meabilized with 0.5% (v/v) Triton X-100 containing 0.1% (w/v) so- dark cytoplasm were considered as surviving oocytes. Only sur- dium citrate for 1 h at RT. The oocytes were then washed again in viving oocytes were transferred to 25-mL drops of the culture me- PBS-PVP solution and incubated in the TUNEL reaction cocktail at dium (1 embryo/mL) covered by 3.5 mL of mineral oil. The culture 38.5 C for 1 h in the dark. Positive and negative control samples medium was synthetic oviduct fluid (Caisson Labs, Smithfield, UT, were included in each assay. Oocytes exposed to DNase I (50 mL of USA) supplemented with 0.96 mg/mL BSA, 88.6 mg/mL sodium py- RQ1 RNase-free Dnase (50 U/mL)) for 1 h at RT served as positive ruvate, 2% (v/v) non-essential amino acids, 1% (v/v) essential amino controls, and oocytes incubated in the absence of the terminal TdT acids, 0.5% gentamicin and 2% (v/v) FBS. Presumptive zygotes were enzyme served as negative controls. After washing in PBS-PVP, incubated at 38.5 C in a humidified 5% CO2 and 5% O2 atmosphere controls and samples were mounted on poly L-lysine-treated cov- for 8 days. Embryo development was recorded at 48 h post- erslips fitted with a self-adhesive reinforcement ring in a 3-mL drop insemination (cleavage) and Days 7 and 8 (blastocysts) post- of Vectashield containing 125 ng/mL of DAPI (Vectorlabs, Burlin- insemination (pi). Day 8 blastocysts were fixed and immuno- game, CA, USA) and flattened with a coverslip. Preparations were stained to assess total cell number (TCN), inner cell mass (ICM) sealed with clear nail varnish and stored at 4 C in the dark until number, trophectoderm (TE) cell number, and apoptosis rate (AR). their observation within the following 2 days. An epifluorescence microscope (Axioscop 40FL; Carl Zeiss, Go € ttingen, Germany) was 2.7. Spindle configuration used to detect TUNEL-positive nuclei (fluorescein isothiocyanate- conjugated TUNEL label; excitation 488 nm; emission 517 nm) After 24 h of IVM, oocytes were completely denuded of cumulus while nuclei were localized using the DAPI filter (DAPI; excitation cells by gentle pipetting before immunostaining for tubulin and 405 nm; emission 460 nm). Nuclei were scored as having either chromatin detection as described previously by García-Martínez intact (TUNEL []; blue stain) or fragmented (TUNEL [þ]; green et al. [21]. Briefly, oocytes were fixed in 2% (w/v) stain) DNA. The percentage of TUNEL-positive oocytes was calcu- paraformaldehydeephosphate buffer saline (PFA-PBS) for 30 min at lated as the ratio between TUNEL[þ] oocytes and the total number 38.5 C. Oocytes were then permeabilized in Triton X-100 (2.5% (v/v) of oocytes analyzed in each group. in PBS) for 20 min and blocked in 3% BSA (w/v) in PBS for 30 min at 38.5 C. The fixed oocytes were incubated with mouse anti-a- 2.9. Differential staining of blastocysts and DNA fragmentation tubulin monoclonal antibody (TU-01, Invitrogen, CA, USA; 1:250 dilution) overnight at 4 C, followed by incubation with the anti- Day 8 bovine blastocysts were immunostained for differential mouse IgG antibody Alexa Fluor™ 488 (Molecular Probes, Paisley, cell counts and apoptosis analysis as previously described by UK; 1:5000) at 38.5 C for 1 h. Oocytes were washed three times in Martinez-Rodero et al. [26] with some modifications. Unless pre-warmed PBS supplemented with 0.005% (v/v) of Triton X-100 otherwise stated, all steps were conducted at 38.5 C. After fixation at 38.5 C for 20 min after each incubation. Groups of 20 oocytes in 2% (v/v) paraformaldehyde diluted in PBS for 15 min, embryos were mounted on poly L-lysine-treated coverslips fitted with a self- were washed three times in PBS. For permeabilization, embryos adhesive reinforcement ring in a 3-mL drop of Vectashield con- were incubated with 0.01% Triton X-100 in PBS supplemented with taining 125 ng/mL 40 ,60 -diamidino-2-phenylindole hydrochloride 5% normal donkey serum (PBS-NDS) for 1 h at RT. Then, the em- (DAPI) (Vysis Inc., Downers Grove, USA) and flattened with a bryos were washed in PBS (  3) and incubated at 4 C overnight coverslip. Preparations were sealed with clear nail varnish and with mouse anti-SOX2 primary antibody (1:100; MA1-014, Invi- stored at 4 C protected from light until observation within the trogen, CA, USA) and rabbit anti-caspase 3 primary antibody (1:250, following 2 days. An epifluorescence microscope (Axioscop 40FL; CST9664S, Cell Signaling Technologies, CA, USA) in a humidified €ttingen, Germany) was used to examine tubulin (Alexa Carl Zeiss, Go chamber. Afterwards, once washed in 0.005% Triton X-100 in PBS- Fluor™ 488; excitation 488 nm; emission 525 nm) and chromatin NDS for 20 min, the embryos were incubated with the secondary 113 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 Fig. 1. Representative confocal laser-scanning photomicrographs of spindle microtubule and chromosome configurations in IVM bovine oocytes after CPA exposure or vitrification. (A) Normal barrel-shaped MII spindle with microtubules forming a clear meiotic spindle with compact chromosomes arranged at the equator of the structure. (B) Abnormal spindle morphology showing partly disorganized chromosomes. (C) Abnormal spindle structures with completely disorganized microtubules and partly disorganized chromosomes. (D) Abnormal spindle structure associated with completely disorganized microtubules and chromosomes. (E) Abnormal spindle structure associated with a disrupted microtubule arrangement and chromosomes appearing condensed. (F) Chromosomes with an aberrant, less condensed appearance. Note the absence of microtubules. Scale bar, 10 mm. Green, tubulin (Alexa Fluor™ 488); blue, chromosomes (DAPI). The white arrowheads indicate polar bodies. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) antibody goat anti-mouse IgG Alexa Fluor™ 568 (1:500; A-11004, MA, USA) for 1 h in a humidified chamber. Embryos were then ThermoFisher, Waltham, MA, USA) and a goat anti-rabbit IgG Alexa washed thoroughly in 0.005% Triton X-100 in PBS-NDS for 5 min, Fluor™ 488 antibody (1:500, A-11001, ThermoFisher, Waltham, mounted on poly-L-lysine treated coverslips fitted with a self- 114 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 adhesive reinforcement ring in a 3-mL drop of Vectashield con- WS2, and HM media at 25 C as previously described; and 2) oo- taining 125 ng/ml DAPI (Vectorlabs, Burlingame, CA, USA), and cytes exposed to ES for 2 min and 30 s followed by exposure to VS, flattened with a slide. The preparation was sealed with clear nail WS2, and HM media at 38.5 C as previously described. In both varnish and stored at 4 C protected from light until observation experimental groups, exposure to WS1 was performed at 38.5 C. A within the following 2 days. Confocal images of 0.5 mm-serial sec- sample of the oocytes in each group was vitrified/warmed using the tions were captured with a confocal laser-scanning microscope Cryotop method and allowed to recover for 2 additional h (VIT25 (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Ger- and VIT35.8, respectively) while the other half were only exposed many) to examine the ICM nucleus (SOX2-Alexa Fluor™ 568; to the vitrification and warming solutions without plunging them excitation 561 nm; emission 603 nm), cell nucleus (DAPI; excitation in liquid nitrogen to assess CPA toxicity (CPA25 and CPA38.5, 405 nm; emission 460 nm) and DNA fragmentation (caspase 3- respectively). Fresh, non-vitrified in vitro matured oocytes served as Alexa Fluor™ 488; excitation 488 nm; emission 525 nm). TCN, ICM the Control group. A sample of oocytes from each of the five cell number, and apoptotic cells were analyzed using Imaris 9.2 treatment groups (Control, CPA25, CPA38.5, VIT25, and VIT38.5) software (Oxford Instruments, UK). Individual nuclei were counted was collected at 24 h of IVM to assess spindle and chromosome and assessed as intact (caspase-3(); blue/red stain) or fragmented configurations (6 replicates) and DNA fragmentation (4 replicates). (caspase-3(þ), green stain) DNA, TE cells (SOX2(OX2(, green stain) In a second trial of experiments, we tested the effect of exposure DNA, TE cells (SOXs, UK) (Fig. 2). The AR was calculated as the ratio of bovine oocytes in vitro matured for 22h to ES to 5 min 30 at 25  C of caspase-3(þ) cells/total number of cells. s or to 2 min 30 s at 38.5  C prior to vitrification/warming on further embryo development. For that purpose, oocytes in the groups 2.10. Experimental design Control, VIT25 and VIT38.5 groups were inseminated and in vitro cultured for 8 days. Cleavage rates were determined at 48 hpi and The experimental design followed in this study is described in blastocysts rates at 168 hpi (day 7; D7) and 192 hpi (day 8; D8) and Fig. 3. After predicting the osmotic response of bovine Metaphase-II are provided as the percentage of zygotes cultured. Early and non- oocytes with a two-parameter permeability model, and reproduc- expanded blastocysts (mid blastocysts) were graded as blastocysts, ing the in silico predictions by in vivo recording the osmometric while expanded, hatching, and hatched blastocysts were termed behavior of bovine oocytes, we limited the duration of the exposure advanced blastocysts [27]. Day 8 blastocysts were fixed and to ES to 5 min 30 at 25  C s or to 2 min 30 s at 38.5  C. In a first trial immunostained to assess TCN, ICM cell number, TE cell number, of experiments, the effect of exposure of IVM bovine oocytes to ES and AR (3 replicates). to 5 min 30 at 25  C s or to 2 min 30 s at 38.5  C followed by vitrification/warming was assessed on spindle and chromosome 2.11. Statistical analysis configurations and DNA fragmentation. To this aim, bovine oocytes in vitro matured for 22 h were randomly assigned to two groups: 1) All statistical tests were performed using R software (V 4.0.3, R oocytes exposed to ES for 5 min 30 s followed by exposure to VS, Core Team, Vienna, Austria). The Kolmogorov-Smirnov test and the Fig. 2. Representative images of D8 non-expanded, expanded, and hatched blastocysts derived from oocytes vitrified/warmed in the 25 C and 38.5 C equilibration protocols. Fluorescence of anti-SOX2 (red) was examined using the Alexa Fluor™ 568 filter to detect ICM cells (B1eB3). Fluorescence of anti-active-caspase-3 antibody (green) was examined by the Alexa Fluor™ 488 filter to detect caspase activity (C1eC3), while DAPI staining (blue) was examined by the DAPI filter for total cell counts (A1-A3). An overlay is provided in (D1-D3). (A1,B1,C1,D1) Non-expanded blastocyst; (A2,B2,C2,D2) Expanded blastocyst; (A3,B3,C3,D3) Hatched blastocyst. Scale bar, 30 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 115 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 Fig. 3. Experimental design. IVM oocytes were randomly assigned to five groups: 1) Control, fresh, non-vitrified oocytes; 2)VIT25: oocytes exposed to ES for 5 min 30 s at 25  C and vitrified/warmed; 3) CPA25: non-vitrified oocytes exposed to ES for 5 min 30 s at 25  C; 4)VIT38.5: oocytes exposed to ES for 2 min 30 s at 38.5  C and vitrified/warmed; and 5) CPA38.5: non-vitrified oocytes exposed to ES for 2 min 30 s at 38.5  C. At 24 h of IVM, oocytes from each of the five treatment groups were denuded and immunostained to assess spindle morphology and DNA fragmentation. Levene test were first used to check the normality of the data and exposed to Me2SO was higher than for oocytes exposed to EG at homogeneity of variance, respectively. When required, data were 25  C (P < 0.05) whereas at 38.5 C no differences in water linearly transformed into the arcsin √x function prior to running permeability were detected between the CPAs. When solute statistical tests. However, if after applying this function, the data permeability was compared between CPAs at each temperature, continued to show a non-normal distribution, they were analyzed only at 38.5 C was Ps for EG higher than for Me2SO (P < 0.05). using a non-parametric test. For the in vitro osmotic behavior experiment, an ANOVA of repeated measures was used, considering 3.1.2. In silico versus in vitro results the given oocyte examined as a random factor. A parametric test For our comparison of the effects of temperature on the cell (one-way ANOVA) was used to examine differences between osmotic response, mature oocytes were exposed to ES containing groups in spindle configuration, DNA fragmentation, and embryo 7.5% Me2SO þ 7.5% EG at the two temperatures (25 C and 38.5 C). development. Non-normally distributed data were analyzed with a As these CPAs are permeable, there is simultaneous water transport non-parametric test (Kruskal-Wallis). For the permeability esti- (because of the osmotic gradient) and CPA influx (because of con- mates and the TCN, ICM, and TE cell number, and AR experiments, a centration gradients) and this produces cell volume changes. two-way ANOVA was used. Secondly, we conducted a pairwise The permeability parameters that we determined using single comparison using the Bonferroni test for parametric analyzes and CPA solutions containing either Me2SO or EG allowed us to make the Wilcoxon test for non-parametric analyzes. Results are predictions for exposure to the ES mixture, which contains both expressed as means ± standard error of the mean (SEM). Signifi- CPAs. Predicted relative volumes and total intracellular solute cance was set at P  0.05. concentrations in oocytes subjected to ES at 25 C or 38.5 C, as a function of time, are shown in Fig. 4. Our model predictions show 3. Results Table 2 3.1. Modeling membrane permeability of bovine MII oocytes Water permeability (Lp) and solute permeability (Ps) of bovine MII oocytes in the presence of 1.55 M Me2SO or 1.55 M EG at 25 C or 38.5 C. 3.1.1. Membrane permeability parameters Temperature n oocytes Membrane permeability parameters Our calculated Lp and Ps for MII bovine oocytes in the presence of CPA Lp (mm/atm  min) Ps (um/s) Me2SO or EG at different temperatures are given in Table 2. Overall,  a,1 Me2SO 25 C 13 1.91 ± 0.13 0.59 ± 0.05a,1 Lp values varied significantly between the temperatures (P < 0.05) 38.5 C 10 2.47 ± 0.18b,1 1.38 ± 0.11b,1 for each CPA. In general, values of Lp for MII oocytes exposed to EG 25 C 14 1.38 ± 0.12a,2 0.78 ± 0.09a,1 1.55 M Me2SO or 1.55 M EG at 25 C were much smaller than for MII 38.5 C 10 2.34 ± 0.16b,1 1.94 ± 0.24b,2 oocytes in the presence of Me2SO or EG at 38.5 C. Moreover, we also found that temperature had a significant effect on the Unless indicated otherwise, data are given as the mean ± SEM. a,b Different super- script letters indicate significant differences between temperatures for the same permeability of solutes in both CPAs (P < 0.05). In general, solute CPA (P < 0.05). 1,2 Different superscript numbers indicate significant differences in permeability increased by a factor of about two as the temperature water or solute permeability between Me2SO and EG CPA for the same temperature increased from 25 C to 38.5 C (Table 2). Also, the Lp for MII oocytes (P < 0.05). Me2SO, dimethyl sulfoxide; EG, ethylene glycol. 116 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 that oocytes exposed to ES shrink to a minimum of 53% of their normally configured spindle between the control group and oo- isotonic volume after 8 s of exposure, whereas at 38.5 C the oocytes cytes exposed or vitrified at 38.5 C. However, oocytes exposed or are predicted to shrink to 60% within 5 s. Moreover, simulations vitrified at 25 C showed a significantly lower proportion of oocytes predict that oocytes at 38.5 C swell back to their isotonic volume with normal spindle and chromosome configurations compared to faster than at 25 C, recovering their original volume after 2.33 min the control group, due primarily to a higher proportion of oocytes or 5 min, respectively. Total intracellular solute molarity is achieved with decondensed or disorganized microtubules or chromosomes faster at 38.5 C than at 25 C. However, in both cases, the solute in the treated groups. concentration gets close to the equilibrium value within the first No significant differences among the treatment groups were minute of exposure to ES (Fig. 4). observed in proportions of oocytes with a disorganized, decon- As in the simulations, the in vitro oocyte osmotic response of densed, or absent spindle configuration, except in the VIT25 group, bovine MII oocytes exposed to ES depicted in Fig. 4 indicates when in which a significantly higher proportion of oocytes featured oocytes attained their minimum volume (Vmin ~61% at 25 C after disorganized spindle and chromosome configurations compared to 15 s and 65% at 38.5 C after 10 s) and when they reach their new the control group. final equilibrium volume (5 min 30 s at 25 C and 2 min 30 s at 38.5 C). As expected, we observed the reduced volume of oocytes in 3.3. Percentages of TUNEL-positive oocytes observed in IVM bovine all groups attributable to water outflow. However, after this initial oocytes vitrified/warmed in the 25 C or 38.5 C equilibration shrinkage, the cells swell as CPA permeates the cell membrane protocols driven by the concentration gradient (Fig. 4). These relative volume changes are dependent on temperature and were in good agree- CPA exposure or vitrification/warming of bovine oocytes after ment with the model predictions. exposure to the equilibration solution for 5 min 30 s at 25 C significantly increased the percentage of oocytes with fragmented 3.2. Spindle configurations observed in IVM bovine oocytes vitrified/ DNA compared to the fresh control group. Although without sig- warmed in the 25 C or 38.5 C equilibration protocols nificance, percentages of TUNEL-positive oocytes in the 38.5 C vitrified group were similar to those in the fresh non-vitrified To assess whether reducing the preparation time of oocytes for treatments (Fig. 5A). Representative images of DNA fragmentation vitrification at a certain temperature would modify oocyte mor- detected by TUNEL analysis are pictured in Fig. 5B. phofunctionality, oocytes were exposed to the ES for 5 min 30 s at 25 C and for 2 min 30 s at 38.5 C and then analyzed for their 3.4. Embryo development in IVM bovine oocytes vitrified/warmed spindle configurations compared to those of non-treated controls. in the 25 C or 38.5 C equilibration protocols In this set of experiments, we used 987 oocytes. Results are sum- marized in Table 3. Table 4 compares the effects on early in vitro embryo develop- Percentages of bovine oocytes reaching the MII stage did not ment of exposing bovine oocytes in vitro matured for 22 h to CPA differ between treatments, except for oocytes exposed to ES at 25 C solutions at 25 C or 38.5 C prior to vitrification/warming. Vitrifi- for 5 min 30 s which showed a significantly lower percentage of cation after exposure of oocytes to CPA solutions at 25 C resulted in nuclear maturation than fresh, non-vitrified oocytes. Neither were significantly lower cleavage rates and D7 and D8 blastocyst yields differences observed in the percentages of oocytes exhibiting a when compared to the control non-vitrified oocyte group. Fig. 4. Comparison between model predictions and in vitro results obtained in bovine MII oocytes exposed for 9 min to ES at 25 C or 38.5 C. Simulation of the osmotic response (solid line) of bovine MII oocytes when transferred to ES and predicted intracellular CPA concentration (dotted line) at 25 C (gray) and 38.5 C (black). The figure also shows experimental volume changes produced in bovine MII oocytes after exposure to ES at 25 C (gray circles) and 38.5 C (black circles). Data are the means of relative volumes ± SEM. Ns: means of relative volumes that do not differ statistically from the volume in isotonic solution. QR code links to a representative time-lapse video for MII bovine oocytes exposed to ES at 25 C or 38.5 C. 117 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 Table 3 Spindle morphology and chromosome alignments observed in IVM bovine oocytes vitrified/warmed in the 25 C or 38.5 C equilibration protocols. Treatment N MII (%) Normal spindle configuration Microtubule distribution (%) Chromosome distribution (%) oocytes (%) Dispersed Decondensed Absent Dispersed Decondensed Absent a Control 227 181 120 (66.69 ± 2.28) 43 15 3 43 18 0 (0.00 ± 0.00) (79.19 ± 1.59)a (22.19 ± 2.00)a (9.53 ± 3.87) (1.59 ± 1.30) (22.19 ± 2.00)a (11.12 ± 3.52) b CPA25 183 109 46 (42.00 ± 2.63) 36 19 8 38 25 0 (0.00 ± 0.00) (62.16 ± 4.26)b (33.21 ± 5.46)ab (16.85 ± 6.27) (7.94 ± 3.75) (34.49 ± 5.31)ab (23.51 ± 4.68) a CPA38.5 211 151 90 (61.16 ± 5.40) 41 14 6 41 20 0 (0.00 ± 0.00) (70.30 ± 2.64)ab (27.54 ± 4.18)a (8.05 ± 2.90) (3.26 ± 1.76) (27.54 ± 4.18)a (11.30 ± 2.98) VIT25 164 110 43 (38.38 ± 1.52)b 50 13 4 50 17 0 (0.00 ± 0.00) (66.54 ± 3.53)ab (45.76 ± 2.61)b (12.79 ± 2.78) (3.07 ± 1.46) (45.76 ± 2.61)b (15.86 ± 3.10) VIT38.5 198 143 72 (53.49 ± 4.00)ab 46 16 9 46 25 0 (0.00 ± 0.00) (71.42 ± 5.22)ab (32.26 ± 1.62)ab (10.19 ± 1.49) (4.07 ± 2.61) (32.26 ± 1.62)ab (14.25 ± 3.91) Unless indicated otherwise, data are given as the mean ± SEM. a,b Within columns, values with different superscript letters differ significantly (P < 0.05). Rates of oocytes with the given morphology were calculated from the total number of oocytes reaching the MII stage. Control, fresh, non-vitrified oocytes; CPA25: non-vitrified oocytes exposed to the equilibration solution at 25 C for 5 min 30 s; CPA38.5: non-vitrified oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s; VIT25: oocytes exposed to the equilibration solution at 25 C for 5 min 30 s and vitrified/warmed; VIT38.5: oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s and vitrified/warmed. Although not significantly different from those obtained after (reviewed by Ref. [3]). The temperature and exposure time of CPAs exposure at 25 C, cleavage rate and percentages of day 7 and day 8 play a crucial role in the whole vitrification process. At higher blastocysts resulting from vitrified oocytes previously exposed to temperatures, oocyte shrinkage is reduced but the potential CPAs at 38.5 C were similar to those obtained from fresh control toxicity of CPAs increases. Besides, each CPA will have an optimal oocytes. Further, the impact of the temperature of exposure to CPAs exposure time and temperature range resulting in minimal cell during vitrification/warming on kinetics blastocyst formation was damage. examined. Vitrification/warming using the 38.5 C protocol tended In this work, based on in vitro observations, we examined the to increase (P  0.1) the proportion of advanced blastocysts used of shorter, dehydration-based protocols at different temper- (expanded, hatching, and hatched) despite no significant differ- atures (25 C vs. 38.5 C) as a first step toward developing an optimal ences detected among the experimental groups (see Table 5). cryopreservation method for MII bovine oocytes. Results indicated that when vitrification was carried out at 38.5 C, reducing the ES 3.5. Differential staining of blastocysts and DNA fragmentation exposure time of MII bovine oocytes to 2 min 30 s gave rise to similar results on spindle morphology, DNA fragmentation and In Table 5 we provide total cell numbers (TCN), numbers of ICM embryo development compared to the used of fresh control oo- cells, numbers of TE cells and apoptotic rates (AR) recorded in Day 8 cytes. When oocytes were exposed to ES for longer time (9 or blastocysts derived from oocytes vitrified/warmed in the 25 C and 10 min) at 38.5 C, apoptosis rates were significantly lower [25,28]. 38.5 C equilibration protocols. Photographs obtained after differ- Hence, the toxicity of CPAs can be reduced by modifying equili- ential cell staining of blastocysts are displayed in Fig. 2. While no bration times and temperatures [29]. Similarly, using the same differences were observed in TCN and numbers of TE cells in methodology a longer ES exposure time (9 or 15 min) to ES at 25 C blastocysts derived from vitrified/warmed oocytes in the 38.5 C results in lower [30] or similar [25,31] cleavage and blastocyst rates protocol, advanced blastocysts were produced with similar TCN to those observed in this study in our VIT25 group yet reduced and TE and ICM cell numbers than those derived from control oo- blastocyst yields have been reported when IVM bovine oocytes are cytes. Although not significantly different from advanced blasto- exposed to ES at 38.5 Ce39 C for 9 or more minutes prior to cysts derived from vitrified/warmed oocytes subjected to the vitrification [21,22,32,33]. Thus, the effects of temperature on 38.5 C protocol, those vitrified/warmed in the 25 C equilibration cytotoxicity are clearly dependent on exposure time [34]. Accord- protocols gave rise to advanced blastocysts with significantly lower ingly, significantly decreased rates of embryo development have ICM cell counts and higher TCN and TE cell counts than blastocysts been described for oocytes overexposed to CPAs at 38.5 C derived from the non-vitrified group. Further, a significant increase [21,22,32,33] while prolonged exposure (9 or 15 min) at 25 C also was observed in TCN, ICM cell numbers, and TE cell numbers as the impaired embryo development [25,31] although showing a much blastocyst stage progressed from blastocyst to advanced blastocyst, less dramatic effect than at 38.5 C. regardless of the treatment group. Although blastocysts derived Rational design approaches combine mathematical models and from vitrified (38.5 C) oocytes showed significantly higher cell biophysical parameters to predict optimized CPA addition and apoptosis rates than those derived from fresh non-vitrified oocytes, removal procedures. As the damage induced by extending cell blastocysts derived from oocytes vitrified in the 25 C protocol volumes beyond osmotic tolerance limits is relatively well under- showed the highest AR compared to the other treatment groups, stood, the most common approach has been to use membrane regardless of blastocyst stage. transport equations and osmotic tolerance limits have been mostly used to predict multi-step procedures able to prevent osmotic 4. Discussion damage [35]. In silico and in vitro analysis of the osmotic behavior of MII oo- Procedures designed to prepare bovine oocytes for vitrification cytes has several limitations. Several assumptions need to be made strive to attain both appropriate intracellular glass-forming con- when calculating the relative volume of an oocyte from a 2D image ditions and prevent an impaired capacity for fertilization and of the shrinking-swelling process, so results may not be accurate development into viable embryos. Because of the possible effects of [36,37]. Also as osmotic behavior is usually determined separately numerous factors during the equilibration stage such as the CPAs for low concentrations of each CPA [38e40] under different employed and their concentrations, as well as the temperature and hyperosmotic CPA conditions, this behavior is likely to vary [41]. For time of exposure to these CPAs, success with bovine oocyte survival example, at CPA concentrations needed for oocyte vitrification, and development has been limited and variable among studies CPAs could affect cytoplasmic phospholipid bilayer permeability 118 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 Fig. 5. TUNEL detection of fragmented oocyte DNA. (A) Effects of exposing IVM bovine oocytes to the equilibration solution for 5 min 30 s at 25 C or 2 min 30 s at 38.5 C before vitrification/warming on percentages of TUNEL-positive oocytes. Data are presented as the mean ± SEM. a,b Values with different letters differ significantly (P < 0.05). Treatment groups: Control: fresh, non-vitrified oocytes; CPA25: non-vitrified oocytes exposed to the equilibration solution at 25 C for 5 min 30 s; CPA38.5: non-vitrified oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s; VIT25: oocytes exposed to the equilibration solution at 25 C for 5 min 30 s and vitrified/warmed; VIT38.5: oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s and vitrified/warmed. Control: n ¼ 106; CPA25: n ¼ 92; CPA38.5: n ¼ 110; VIT25: n ¼ 164 and VIT38.5: n ¼ 175 oocytes. (B) Representative images of IVM bovine oocytes stained with TUNEL ((i1,i2); green) and DAPI ((ii1,ii2); blue). (i1,ii1): TUNEL-positive oocyte; (i2,ii2): TUNEL-negative oocyte. An overlay is given in (iii1,iii2). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) [42]. Further, the biophysical parameters of individual oocytes also interval intracellular water efflux is close to completion and the seem to be highly variable [43]. Thus, permeability models and permeation of low molecular weight CPAs leads to similar intra- in vitro observations may not accurately reflect the physiological cellular and extracellular solute concentrations. Thus, lengthening situation. exposure to CPA solutions does not improve cytosolic glass for- Despite these limitations, our in silico and in vitro results were mation. This means that rather than the usual 9e15 min of CPA fairly consistent. The time required for the oocytes to reach the exposure time used for bovine oocytes, a better approach could be equilibrium cell volume upon exposure to standard ES increased as oocyte exposure to non-vitrifying hypertonic solutions just until the temperature decreased, such that 2 min 30 s was needed at the equilibrium cell volume is reached to prepare them for vitrifi- 38.5 C and 5 min 30 s at 25 C. This is important because during this cation. In the present study, we modified the equilibration protocol 119 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 Table 4 Developmental competence of embryos derived from IVM bovine oocytes vitrified/warmed after the 25 C or 38.5 C equilibration protocol. n Cleavage rate D7 blastocysts D8 blastocysts nD8 D8 blastocysts Blastocysts Advanced a a a a Control 170 141 (83.06 ± 2.07) 49 (28.17 ± 3.34) 60 (33.78 ± 5.07) 60 31 (17.44 ± 1.89) 29 (16.33 ± 3.90) VIT25 158 86 (55.18 ± 5.09)b 19 (12.35 ± 2.54)b 20 (13.23 ± 1.81)b 20 11(7.31 ± 1.40)b 9 (5.92 ± 2.19) VIT38.5 96 62 (64.92 ± 6.54)ab 18 (18.83 ± 2.23)ab 22 (23.94 ± 4.88)ab 22 10 (10.38 ± 2.71)ab 12 (13.56 ± 2.42) Unless indicated otherwise, data are given as the mean ± SEM. a,b Within columns, values with different superscript letters differ significantly (P < 0.05). Cleavage rates (48 hpi) and D7 and D8 blastocyst yields were based on the number of oocytes that survived at Day 1 post-insemination. nD8: total number of blastocysts observed at day 8 post- insemination. Oocyte survival was assessed on the basis of integrity of the oocyte membrane and zona pellucida, along with discoloration of the cytoplasm. Blastocysts: early and non-expanded blastocysts (intermediate-stage blastocysts); Advanced: expanded, hatching, and hatched blastocysts. Control: embryos derived from fresh, non- vitrified oocytes; VIT25: embryos derived from oocytes exposed to the equilibration solution at 25 C for 5 min 30 s and vitrified/warmed; VIT38.5: embryos derived from oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s and vitrified/warmed. of a new vitrification/warming method designed to represent the here at 38.5 C [21,22]. This may indicate that IVM bovine oocytes current standard practice to prepare bovine oocytes [14] according are more sensitive to the temperature of exposure to ES and VS than to the in vitro osmotic behavior of oocytes at different tempera- to the detrimental effects of CPA toxicity. tures. These modifications to the protocol were validated by In this study, vitrification after CPA exposure at 38.5 C for 2 min examining oocyte spindle status, oocyte DNA fragmentation and 30 s did not significantly affect the occurrence of DNA fragmenta- further embryo development after vitrification/warming of MII tion in oocytes when compared to fresh controls. Contrarily, bovine oocytes. exposure or vitrification of oocytes at 25 C significantly increased The main hurdle in developing successful protocols for the the percentage of DNA fragmentation after warming. Some authors cryopreservation of mammalian oocytes is preserving the integrity have questioned whether CPA toxicity is related to the osmotic of the meiotic spindle when oocytes are cooled below physiological stress of adding and removing CPAs. Although osmotic stress can temperature. Temperature fluctuations directly affect the cyto- indeed cause cytotoxicity, experimental evidence suggests that skeletal and microtubular systems of mature bovine [6] and human CPAs are more chemically toxic [49]. While the mechanisms of CPA [44] oocytes. While the effects of cooling on the spindle seem toxicity remain to be clearly established [50], hydrophobic in- reversible in the mouse oocyte, with normal spindle formation teractions between CPAs and proteins, and a longer lifetime of occurring after step-wise re-warming [45], bovine and human CPAewater hydrogen bonds [51] have been proposed. These agents oocytes are less resilient because of the irreversibility of have also been accused of varying intracellular pH [52], increasing temperature-induced spindle disruption [6,44,46]. In our study, intracellular calcium contents [53], and causing formaldehyde exposure of oocytes to CPAs at 25 C for 5 min 30 s yielded the formation in cryopreservation solutions [54] (or reviewed by lowest percentage of oocytes reaching the MII stage. Aman and Ref. [50]). Parks [6] reported that the spindles of MII bovine oocytes started to The results of our embryo development experiments designed depolymerize when cooled to 25 C for 1 min or more, and the to assess oocyte competence after vitrification/warming indicate extent of depolymerization (or disassembly) was also directly comparable cleavage and blastocyst rates of vitrified bovine oocytes related to CPA exposure duration [6]. We also observed that CPA after exposure to CPA solutions at 38.5 C to those of control fresh exposure or vitrification after equilibration at 25 C for 5 min 30 s oocytes. Moreover, although not significant, shorter CPA exposures also resulted in reduced proportions of oocytes displaying normal at 38.5 C gave rise to almost double the percentage of blastocysts spindle configurations. Other authors have described higher pro- obtained after longer CPA exposure at 25 C. Our results clearly portions of spindle alterations in MII bovine oocytes vitrified after indicate the importance of temperature and time of CPA addition similar or longer exposures times to the equilibration solution at prior to vitrification. Cytotoxicity has been shown to increase with 25 C [47,48] when compared to their fresh counterparts. However, increased temperatures for relatively long exposure times [34,55]. no significant increase was noted here in chromosomal abnor- This has led to the general assumption that CPA exposure should be malities after CPA exposure at 38.5 C for 2 min 30 s before vitrifi- carried out at low temperatures (25 C). However, the temperature cation, suggesting that the bovine oocytes were able to recover of addition significantly affects the time needed to achieve full from cooling or CPA-induced damage to the oocytes' spindle mi- equilibration. At a higher temperature, equilibration occurs much crotubules. In prior work, we observed that bovine oocytes vitrified more quickly, allowing for shorter incubation times and reducing after 10 min of exposure to ES at 38.5 C led to similar rates of cell exposure to CPAs during their addition and removal. Due to maturation and normal spindle configurations to those detected faster mass transport across the cell membrane at 38.5 C, Table 5 Total cell numbers, number of cells in the ICM and TE, and rate of apoptotic cells recorded in Day 8 blastocysts derived from oocytes vitrified/warmed after the 25 C and 38.5 C equilibration protocols. Day 8 blastocysts n TCN ±SEM ICM cell number ±SEM TE cell number ±SEM AR± SEM Blast Adv Blast Adv Blast Adv Blast Adv Blast Adv Control 31 29 95.2 ± 2.21 155.7 ± 5.6a,2 12.5 ± 0.7a,1 24.3 ± 1.0a,2 82.7 ± 2.11 131.45 ± 5.2a,2 6.8 ± 0.4a,1 6.0 ± 0.5a,1 VIT25 11 9 87.8 ± 3.21 180.5 ± 5.4b,2 8.9 ± 0.5b,1 18.5 ± 2.9b,2 79.0 ± 3.11 162.0 ± 6.2b,2 19.9 ± 3.1b,1 15.3 ± 1.4b,2 VIT38.5 10 12 92.0 ± 4.51 162.8 ± 8.1ab,2 10.8 ± 1.9ab,1 20.5 ± 2.3ab,2 81.1 ± 4.21 142.3 ± 7.9ab,2 13.3 ± 1.3c,1 10.3 ± 1.0c,1 Unless indicated otherwise, data are given as mean ± SEM. a,b,c Values within columns with different superscripts differ significantly (P < 0.05); 1,2 Values within rows with different superscripts differ significantly (P < 0.05). Blast: early and non-expanded blastocysts (intermediate stage blastocysts); Adv: expanded, hatching, and hatched blastocysts. TCN: total cell number; ICM: inner cell mass; TE: trophectoderm; AR: apoptosis rate. Control: embryos derived from fresh, non-vitrified oocytes. VIT25: embryos derived from oocytes exposed to the equilibration solution at 25 C for 5 min 30 s and vitrified/warmed. VIT38.5: embryos derived from oocytes exposed to the equilibration solution at 38.5 C for 2 min 30 s and vitrified/warmed. 120 T. García-Martínez, I. Martínez-Rodero, J. Roncero-Carol et al. Theriogenology 184 (2022) 110e123 equilibration of the oocyte occurs earlier than at 25 C and less time indicate that limiting the exposure time to ES to 2 min 30 s at 38.5 C is required for CPA addition. If we bear in mind that CPA exposure at seems to improve the quality of vitrified/warmed oocytes 38.5 C had fewer deleterious effects on the meiotic spindle and improving blastocyst rates and quality by protecting spindle oocyte apoptosis, CPA exposure at 38.5 C for 2 min 30 s may have integrity and reducing DNA fragmentation. These data also indicate allowed the oocyte to achieve the minimum intracellular glass- that toxic damage at a given temperature may be reduced by forming conditions so as not to fully impair the ability of vitri- minimizing the duration of CPA addition and removal while fied/warmed oocytes to be successfully fertilized and develop into maintaining cell volumes within osmotic tolerance limits. Our viable embryos. findings also pave the way for using a similar approach to improve It has been well established that for embryo survival and post- the vitrification of other mammalian oocytes. implantation development, blastocyst cells need to be optimally allocated to the ICM and TE. Studies have shown that a defined Funding minimum number of ICM cells correlates strongly with normal fetal development and an adequate ICM:TE cell ratio seems essential for This study was founded by Spanish Ministry of Science and embryo viability and pregnancy [56]. Moreover, excessive alloca- Innovation (Project PID2020-116531RB-I00) and the Generalitat de tion of cells to the TE possibly leads to early pregnancy loss [56,57]. Catalunya (Project No. 2017 SGR 1229). Scholarship to Mrs. Tania Our blastocyst kinetics experiments revealed a clear trend towards García-Martínez (2019 FI_B2 00055). a higher hatching ability of blastocysts derived from oocytes vitri- fied/warmed in the 38.5 C protocol. Further, blastocysts in the Availability of data and materials 38.5 C vitrification group featured similar TCN, ICM, and TE cell counts as in the non-vitrified groups, regardless of blastocyst stage. All data generated or analyzed during this study are included in In contrast, higher TE and apoptotic cell numbers and lower ICM the published article. cell counts were observed in blastocysts derived from oocytes vitrified at 25 C. While in preimplantation embryos apoptosis CRediT authorship contribution statement serves to eliminate compromised cells, a high incidence of apoptosis results in a morphologically abnormal embryo [58]. This Tania García-Martínez: Conceptualization, Formal analysis, may explain why the lower apoptosis rate observed here in blas- Investigation, Methodology, Writing e original draft, All authors tocysts derived from oocytes vitrified/warmed in the 38.5 C pro- have read and agreed to the published version of the manuscript. tocol gave rise to higher (P < 0.1) percentages of advanced Iris Martínez-Rodero: Investigation, Methodology, All authors blastocysts. have read and agreed to the published version of the manuscript. At this point we should mention that most human oocytes are Joan Roncero-Carol: Investigation, Methodology, All authors have vitrified by long exposure (up to 8e15 min) to equilibration media read and agreed to the published version of the manuscript. Iva n and using similar CPA concentrations to those used in our study at Yanez-Ortiz: Methodology, All authors have read and agreed to the room temperature (25 C) [14,59e62]. This protocol has proved to published version of the manuscript. Adam Z. Higgins: Conceptu- be the best strategy for the cryopreservation of all human embryo alization, Formal analysis, Methodology, Supervision, Validation, developmental stages, especially of mature oocytes, as it signifi- Writing e review & editing, All authors have read and agreed to the cantly improves cryosurvival and clinical outcomes when published version of the manuscript. Teresa Mogas: Conceptuali- compared to slow freezing, and has also allowed for the creation of zation, Formal analysis, Funding acquisition, Project administra- oocyte banks [63]. Our worse results using a longer equilibration tion, Resources, Supervision, Validation, Writing e review & time at 25 C in bovine oocytes compared with the vast amount of editing, All authors have read and agreed to the published version data supporting this vitrification protocol for human oocytes, il- of the manuscript. lustrates that factors other than CPA exposure times and temper- atures may have an impact on oocyte cryosurvival (for a review, see Declaration of competing interest Ref. [3]). Beside other features, we propose that the different out- comes observed for bovine and human oocytes vitrified using the The authors declare no conflict of interest. same procedure might be related to differences in oocyte mem- brane permeability or the accumulation of lipid vesicles in the Acknowledgments ooplasm [64]. In effect, it is well-known that the presence of these intracytoplasmic lipid droplets is an obstacle for the successful Not applicable. cryopreservation of bovine oocytes [65]. Nevertheless, shorter protocols for the vitrification of human oocytes have been recently References proposed [66,67]. This trend allows for more efficient IVF workflow while minimizing the impacts of CPA toxicity. [1] Prentice JR, Anzar M. Cryopreservation of Mammalian oocyte for conservation of animal genetics. Vet Med Int 2010;2011. [2] Mogas T. Update on the vitrification of bovine oocytes and invitro-produced 5. Conclusion embryos. Reprod Fertil Dev 2018;31:105e17. [3] Dujickova L, Makarevich AV, Olexikova L, Kubovicova E, Strejcek F. Method- In conclusion, in this study we validate new CPA equilibration ological approaches for vitrification of bovine oocytes. Zygote 2020:1e11. [4] Fahy GM. Cryoprotectant toxicity neutralization. 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